FLAVONOIDS OF HAPLOPAPPUS SCROBICULATUS A N D HAPLOPAPPUS SERICEUS NEZHUN ATES and AYHAKULUBELEY Faculty of Phnrmcicy, Cniiiersity of Istunbul, Istanbul, T u r k e y and W. DENNISCLARKand GREGORY K. BRONK Department of Botany and Microbiology, A rizona State Cninlersity, T e m p e , Arizona and TOMJ . MABRY Department of Botany, T h e Ilnirtersity of Texas at A u s t i n , A u s t i n , Texas and G . DELLAMOSICA and J. CHOPIN Laboratory of Biological Chemistry, C nirtersity Claude Bernard, 69622 Villeurbunne, France
ABSTRACT.-Thirteen flavonoids and the coumarin esculetin were isolated from H a p l o p a p p u s scrobiculatus, and five of the flavonoids were also found in H . sericeus. Both species yielded quercetin, quercetin 3-&D-glucoside, isovitexin, vitexin, and vicenin-2 (6,8-di-C-glucosylapigenin). I n addition, H . scrobiculatus was found t o accumulate isorhamnetin, isorhamnet in %@D-glucoside, 6-methoxyluteolin 4I-methyl ether, 6-methoxyluteolin 7-methyl ether, 6methoxyluteolin, kaempferol 7-methyl ether, and isoschaftoside (6-Carabinosyl-8-Cglucosylapigenin).
We have previously reported Haplopappus flavonoids from H . canescens (1) of section Haplopappus, H . rengifoanus ( 2 ) and H . joliosus ( 3 ) of section Polyphylla, and H . integerrimus var. punctatus (4)of section Steriphe. Sections Haplopappus and Steriphe are closely related and are perhaps the only true Haplopappus in South America (5). We report here further studies from a continuing investigation of the systematic relationships between these two sections and between South American Haplopappus and its Sorth American relatives. Of note in this study is the accumulation of C-glucosylflavones in both H . scrobiculatus (Sees) DC. (section Haplopappus) and H . sericeus Phil. (section Steriphe), compounds which have not previously been found in South American species but which are common in closely related Sorth American taxa (6).
EXPERIJIENTAL' PLAKT MATERIAL.-Leaves of H . scrobiculatus were collected in Chile, 5 km west of Portillo, Prov. Aconcagua, (Clark and Brown 1337, 1338) and on the outskirts of Farellones, Prov. Santiago, (Clark & Brown 1351) in January and February 1979; those of H . sericeus (Clark & Brown 1347) were collected on the outskirts of La Parva, Prov. Santiago, in February 1979. Voucher specimens are deposited in the Herbarium at Arizona State University. EXTRACTION A S D ISOLATION OF THE FLAF OSOIDS.-A~~of the general chromatographic techniques employed were described in the previous report on the flavonoids of H . rengifoanus (2). Powdered leaves of H . scrobiculatus (Xos. 1337, and 1338,250 g combined; No. 1351,400 g) and H . sericeus (237 g) were separately extracted in a Soxhlet sequentially with petroleum ether (bp 30-60"), chloroform, ethyl acetate, and ethyl alcohol. The flavonoids from H . scrobiculatus were found in the ethyl acetate and alcohol extracts, while in H . sericeus they were found only in the alcohol extract. Since two-dimensional paper chromatography showed the same flavonoids in the ethyl acetate extracts of each of the collections of H . scrobiculatus, they were combined. The combined concentrate (10 g) was chromatographed over a Polyclar column (4 x 50 cm). The column was eluted with Egger's solvent chloroform-methanol-methyl ethyl ketone-acetone (4:2:0.5:0.1); the polarity of the eluate was increased by reducing the percentage of chloroform. All of the compounds from the Polyclar column were cleaned over Sephadex LH-20. The Polyclar column yielded the following compounds: 6methoxyluteolin 4'-methyl ether (5 mg), 6-methoxyluteolin 7-methyl ether (4 mg), 6-methoxyluteolin (3 mg), and kaempferol 7-methyl ether. The combined alcohol concentrate (10 g) of H . scrobiculatus was chromatographed over a Polyclar column (5 x 50 cm). The elution was initiated with 'Spectra were recorded with the following instruments: uv Varian Techtron model 635; pmr Varian 90 MHz; ms DuPont 21-491 and AEI 902, direct inlet a t 70 eV. Adsorbents for the tlc and cc were from E. hlerck, Macharey-Nagel and GAF Corp; Sephadex LH-20 was from Pharmacia. 189
Journal of Natural Products
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[T'ol. 45,
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ethanol, and the polarity was increased by the gradual addition of water up to lWr;. The following compounds were obtained: isorhamnetin (G mg), isorhamnetin 3-@-l)-glricoside (5 mg), quercetin (5 mg), esculetin (5 mg), isovitevin (8 mg), vitexin (2 mg), and a mixture (8 mg) of vicenin-2 and a G,S-di-Cglucosyl compound with a luteolin skeleton (these two compounds were separated on prep tlc plates after permethylation of the miuture). Next, 7 mg of isoschaftoside (GC-arabinosyl-8-C-glucosylapigenin)with traces of vicenin-2 were obtained. These two compounds were also separated on prep tlc plates after permethylation. Finally, the column yielded quercetin S-&D-glucoside (4 mg). The ethanol extract (yield 7 g of concentrate) of Haplopuppus sericeus was also chromatographed on a Polyclar column. The column was eluted with ethyl alcohol with gradually increasing percentage of water up to 1 0 0 ~ c . Quercetin (7 mg), quercetin 3-@-I)-glucoside (12 mg), vicenin-2 (4 mg), isoviteuin (3 mg) and vitexin (3 mg) were obtained. QUERCETIN, ISORHAM N E T I N , KAEMPI.FROL 7-METHYL ETHER, GMETHOXYLUTEOLIN, &WETHOXI-4'-METHYL ETHER, 6-METHOXTLUTEOLIN 7-METHYL ETHER-UV, ms, and tlC comparisons with standard samples established the structures of these compounds. LUTEOLIN
QUERCETIN 3-p-D-GLL-COSIDE, ISORHAMNETIN
[email protected] of these compounds with acid (0.1 N TFA) and @-glucosidase yielded the expected aglycones, quercetin and isorhammetin (uv and tlc comparison) and glucose (tlc comparison). Uv spectral d a t a and colors under uv light (366 nm) before and after hydrolysis, tlc comparison with authentic samples, and uv spectral d a t a established the structures of these two glucosides.
ISOVITEXIN, VITEXIN.-UV, ms and standard sample comparison were employed to establish the structures of these compounds. TICEXIN-2 A N D A N UNIDENTIFIED 6,8-DI-C-GLUCOSYLbLAVONE \\ ITH A LCTEOLIN SKELETON.T h e uv spectral shifts of this fraction indicated that the major compound (by far) must have an apigenin skeleton. It gave the following spectral data: uv X max (MeOH): 332, 272; NaOMe 399 (higher int.), 330,283; AIC13 and AlCl,/HCI 385 (sh), 353,307,262 (sh); NaOAc 368, 296 (sh), 282; NaOAc/H3B03415 (sh), 340 (sh), 321, 278. Although the components of this mixture could not be separated by chromatography on Polyclar, permethylation and prep tlc (Si gel) developed with chloroform-ethyl acetate-acetone (5:4:1) gave two major compounds ( R f 0.22 and 0.33). The ms of the latter was t h a t of a permethyl 6,S-di-C-hexsylapigenin (7) and showed the same R f as P M 6,S-di-Cglucosylapigenin; ms m / z (re1 int.): M+748 (9), 733 (29), 717 (loo), 645 (ll),585 (291, 573 (48). The lower band showed the same R f as P M 6,8-di-C-glucosylluteolin, and the ms confirmed the P M 6,S-di-C-hexosylluteolin structure (7); ms m / z (re1 int.) M+778 (13), 763 (25), 747 (loo), 675 (15), 615 (28), 603 (42). Therefore, this latter natural product must be based on a luteolin or luteolin methyl ether skeleton.
+
+
+
+
IsoScmFTosIDE.-The uv spectrum was identical t o that of agipenin. T h e light yellow color with diazotized benzidine reagent, as well as the R f value in 57, acetic acid (0.43), suggested a G,€&di-C-glycosylapigenin. TICof the P M derivative with standard P M isoschaftoside indicated t h a t the natural product was isoschaftoside; the tlc also showed P M vicenin-2 to be present as a minor constituent. uv and i r spectra as well as standard sample comparison proved i t s
EScuLETIN.-The structure.
ACKNOWLEDGMENTS T h e work undertaken a t Arizona State University was supported by the National Science Foundation (Grant No. DEB-7823897), a t the University of Texas by the Robert A. Welch Foundation (Grant No. F-130), and at the University of Istanbul by the Faculty of Pharmacy. Received 67 July 1981 LITERATURE C I T E D 1. S. Oksuz, A. Ulubelen, W. D. Clark, G. K. Brown and T . J. Mabry, Reo. Lationonmer. Quim., 12, 12 (1981). 2. A. Ulubelen, W. D. Clark, G. K. Brown and T. J. Mabry, J . Nut. Prod., 44, 294 (1981). 3. A. Ulubelen, E. Ayanoglu, W. I). Clark, G. K . Brown and T. J. Mabry, J . Nat. Prod. (in Ulubelen, W. I>. Clark, G . K. Brown, It. R. Kerr and T. J. Mabry, Phyto-
Systematics n;2d Ecology, 8, 257 (1980). 7. M . L. Bouillant, J. Favre-Bonvin and J. Chopin, Phytochemistry, 14, 2267 (1975).
